Single Cell Sequencing Reveals Glial Specific Responses to Tissue Processing & Enzymatic Dissociation in Mice and Humans

Abstract

A key aspect of nearly all single cell experiments is the necessity to dissociate intact tissues into single cell suspensions for processing. While many protocols have been optimized for optimal cell yield, they have often overlooked the effects that dissociation can have on ex vivo gene expression changes during this process. Microglia, the brain’s resident macrophages, are a highly dynamic population that are extremely sensitive to their microenvironment and have been shown to dramatically alter their transcriptome upon stimulation. We demonstrate that use of enzymatic dissociation methods on mouse central nervous system (CNS) tissue induces an aberrant gene expression signature in microglia that can significantly confound downstream analysis. To minimize this issue, we developed a flexible protocol, that can be used with existing enzymatic protocols for fresh tissue, to eliminate artifactual gene expression while allowing for increased cell type diversity and yield. We demonstrate efficacy of this protocol in analysis of diverse CNS cell types and sorted myeloid populations while using enzymatic dissociation. Generation of new and reanalysis of previously published human brain single nucleus RNAseq (snRNA-seq) datasets reveal that a similar signature is also present in post-mortem tissue. Through novel snRNA-seq analysis of acutely-resected neurosurgical tissue we demonstrate that this signature can be induced in human tissue due to technical differences in sample processing. These results provide key insight into the potential confounds of enzymatic digestion and provide a solution to allow for enzymatic digestion for scRNA-seq while avoiding ex vivo transcriptional artifacts. Analysis of human tissue reveals potential for artifacts in current and future snRNA-seq datasets that will require deeper analysis and careful consideration to separate true biology from artifacts related to post-mortem processes.

Minimizing the ex vivo confounds of cell-isolation techniques on transcriptomic -profiles of purified microglia

“…We observed higher levels of cytokine and chemokine transcripts across all Cell-and Sort-TRAP groups when compared to Tissue-TRAP (One-way ANOVA, BHMTC, SNK FDR<0.1, |FC|>2). In an effort to cross-validate our finding with previous studies, we intersected ex vivo microglial activational gene lists from three previous studies (Ayata et al, 2018;Marsh et al, 2020) and identified 21 ex vivo activational transcripts represented in at least two of the studies (Figure 4J; Supplemental Table 5). PCA of the TRAP data from the present study on the 21 ex vivo activational genes shows clear separation of Tissue-TRAP from all other groups in the first component (92.8% explained variance) (Figure 4K).…”

“…In recent years, several studies have suggested that cell-isolation methods cause ex vivo activational effects in microglia (Ayata et al, 2018;Marsh et al, 2020). In this section, our goal was to determine if different sorting techniques result in different levels of ex vivo activation.…”

“…Transcription and translation inhibitors were included during cell preparation, as previously described (Marsh et al, 2020) with slight modifications. Briefly, Actinomycin D (#A1410, Sigma-Aldrich) was reconstituted in DMSO to a concentration of 5mg/mL before being aliquoted and stored at -20°C protected from light.…”

Aging-associated deficit in CCR7 is linked to worsened glymphatic function, cognition, neuroinflammation, and β-amyloid pathology

“…Cluster annotation was performed manually on the basis of the expression of canonical markers. Cells not of interest to the analysis were removed on the basis of these cluster annotations, including oligodendrocytes, ependymal cells, choroid plexus endothelial cells, a small population of pericytes and smooth muscle cells, and a subcluster of microglia with a similar gene signature to artificially ex vivo activated microglia (59). Transcripts were then rescaled, reclustered, and annotated again using the methods described above, and clusters were collapsed on the basis of common cell types.…”

Spatial cell type mapping of the oligodendrocyte lineage in the mouse juvenile and adult CNS with in situ sequencing

“…Many of these genes can be upregulated upon neuronal activation, but also upon stress and apoptosis (Hrvatin et al, 2018). However, stress and apoptosis can be induced by the processing of tissue in single cell transcriptomic approaches (Brink et al, 2017; Marsh et al, 2020). Thus, it is challenging to determine whether stress/activated related signatures are truly biological or artefactual.…”